Large-scale transfection of Chinese Hamster Ovary (CHO) cells with cost-effective reagents for the production of r-proteins suffers from low efficiency and low productivity. In addition, plasmid vectors used in CHO cells are not fully optimized for transient gene expression.
There are some very efficient and commercially available cationic lipids formulation that can be used to transfect CHO cells in serum-free medium, for example FreestyleMax™ from Invitrogen. However, these cationic lipids are very expensive. Also, to improve productivity, it is becoming current practice to lower the cultivation temperature following transfection to prolong the production phase and to enhance productivity. This temperature shift is not “user friendly” when working at large-scale or when using non-refrigerated culture devices (Wave bioreactors, etc). Also, the exact temperature at which the shifts are done may be critical for getting optimal enhancement (e.g. 29 vs. 30 vs. 31 vs. 32 degrees Celsius).
International patent publication WO 2007/048601 reports an expression system in CHO cells stably expressing EBNA1 for the production of r-proteins. However, this document specifically admonishes that the cell lines shall not contain a functional copy of the Epstein-Barr virus (EBV) oriP sequence. Further, the full length EBNA1 structural gene encoding a full length EBNA1 protein is transfected into the cell line, and the oriP sequence is never in the same vector as the EBNA1 gene construct.
International patent publication WO 2002/090533 describes enhanced production of recombinant proteins by transient transfection of suspension-growing mammalian cells. However, only full length EBNA1 structural genes are used encoding full length EBNA1 proteins and only transient expression of a gene of interest is achieved.
International patent publication 2006/096989 describes expression vectors for enhanced transient gene expression and mammalian cells expressing them. However, only HEK293 cell lines are exemplified and the expression system used does not contain both the EBNA1 gene construct and the oriP sequence in the same vector. Further, only transient expression of a gene of interest is achieved.
There is a need in the art for processes, vectors and engineered cell lines for more efficient and productive transfection of cells at a large scale.